Efficient base editing in tomato using a highly expressed transient system
Efficient base editing in tomato using a highly expressed transient system
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DOI：
10.1007/s00299-021-02662-z
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发表时间：
2021-02
影响因子：
6.2
通讯作者：
Shaoze Yuan;Shunsuke Kawasaki;Islam M. Y. Abdellatif;K. Nishida;A. Kondo;T. Ariizumi;H. Ezura;K. Miura
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作者：
Shaoze Yuan;Shunsuke Kawasaki;Islam M. Y. Abdellatif;K. Nishida;A. Kondo;T. Ariizumi;H. Ezura;K. Miura
摘要茄科植物，特别是番茄(Solanum Lycopsicum)，在世界范围内具有巨大的经济价值。番茄是研究果实成熟相关基因功能的独特模式植物。加深对西红柿的了解对植物研究和经济都非常重要。基因组编辑技术，如CRISPR/Cas9，已被用于功能遗传学研究。然而，这项技术仍然存在一些挑战，如转换效率低。此外，必须去除外源的Cas9和gRNA表达盒才能获得空分离株。在本研究中，我们使用了一种高水平的瞬时表达系统来改进碱基编辑技术。先前已经利用双生病毒复制和双终止子建立了高水平的瞬时表达系统。将pBYR2HS载体用于该瞬时表达系统。将nCas9-CDA和sgRNA-slHWS导入到该载体中，通过农渗法在番茄组织中瞬时表达蛋白质和RNA。通过碱基编辑得到的纯合突变体在下一代获得了约18%的效率，无nCas9的下一代植株的效率为71%。下一代所有纯合的碱基编辑植物都不含Cas9。这些发现表明，高水平的瞬时表达系统对番茄的碱基编辑是有用的。
Key messageBase editing in tomatoes was achieved by transient expression.AbstractThe Solanaceae plants, particularly the tomato (Solanum lycopersicum), is of huge economic value worldwide. The tomato is a unique model plant for studying the functions of genes related to fruit ripening. Deeper understanding of tomatoes is of great importance for both plant research and the economy. Genome editing technology, such as CRISPR/Cas9, has been used for functional genetic research. However, some challenges, such as low transformation efficiency, remain with this technology. Moreover, the foreign Cas9 and gRNA expression cassettes must be removed to obtain null-segregants In this study, we used a high-level transient expression system to improve the base editing technology. A high-level transient expression system has been established previously using geminiviral replication and a double terminator. The pBYR2HS vector was used for this transient expression system. nCas9-CDA and sgRNA-SlHWSwere introduced into this vector, and the protein and RNA were then transiently expressed in tomato tissues by agroinfiltration. The homozygous mutant produced by base editing was obtained in the next generation with an efficiency of about 18%.nCas9-free next-generation plants were 71%. All the homozygous base-edited plants in next generation arenCas9-free. These findings show that the high-level transient expression system is useful for base editing in tomatoes.